The ontogeny of hepatic uridine diphosphate-glucuronosyltransferases (UGTs) was investigated by determining their protein abundance in human liver microsomes isolated from 136 pediatric (0-18 years) and 35 adult (age >18 years) donors using liquid chromatography / tandem mass spectrometry (LC-MS/MS) proteomics. Microsomal protein abundances of UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 increased by ∼8, 55, 35, 33, 8, and 3-fold from neonates to adults, respectively. The estimated age at which 50% of the adult protein abundance is observed for these UGT isoforms was between 2.6-10.3 years. Measured in vitro activity was generally consistent with the protein data. UGT1A1 protein abundance was associated with multiple single nucleotide polymorphisms exhibiting noticeable ontogeny-genotype interplay. UGT2B15 rs1902023 (*2) was associated with decreased protein activity without any change in protein abundance. Taken together, these data are invaluable to facilitate the prediction of drug disposition in children using physiologically based pharmacokinetic modeling as demonstrated here for zidovudine and morphine.

No abstract

Huntington’s disease is characterized by a complex and heterogeneous pathogenic profile. Studies have shown that disturbance in lipid homeostasis may represent a critical determinant in the progression of several neurodegenerative disorders. The recognition of perturbed lipid metabolism is only recently becoming evident in HD. In order to provide more insight into the nature of such a perturbation and into the effect its modulation may have in HD pathology, we investigated the metabolism of Sphingosine-1-phosphate (S1P), one of the most important bioactive lipids, in both animal models and patient samples. Here, we demonstrated that S1P metabolism is significantly disrupted in HD even at early stage of the disease and importantly, we revealed that such a dysfunction represents a common denominator among multiple disease models ranging from cells to humans through mouse models. Interestingly, the in vitro anti-apoptotic and the pro-survival actions seen after modulation of S1P-metabolizing enzymes allows this axis to emerge as a new druggable target and unfolds its promising therapeutic potential for the development of more effective and targeted interventions against this incurable condition.

The major objective of this study was to investigate the association of genetic and nongenetic factors with variability in protein abundance and in vitro activity of the androgen-metabolizing enzyme UGT2B17 in human liver microsomes ( = 455). UGT2B17 abundance was quantified by liquid chromatography-tandem mass spectrometry proteomics, and enzyme activity was determined by using testosterone and dihydrotestosterone as in vitro probe substrates. Genotyping or gene resequencing and mRNA expression were also evaluated. Multivariate analysis was used to test the association of UGT2B17 copy number variation, single nucleotide polymorphisms (SNPs), age, and sex with its mRNA expression, abundance, and activity. UGT2B17 gene copy number and SNPs (rs7436962, rs9996186, rs28374627, and rs4860305) were associated with gene expression, protein levels, and androgen glucuronidation rates in a gene dose-dependent manner. UGT2B17 protein (mean ± S.D. picomoles per milligram of microsomal protein) is sparsely expressed in children younger than 9 years (0.12 ± 0.24 years) but profoundly increases from age 9 years to adults (∼10-fold) with ∼2.6-fold greater abundance in males than in females (1.2 vs. 0.47). Association of androgen glucuronidation with UGT2B15 abundance was observed only in the low UGT2B17 expressers. These data can be used to predict variability in the metabolism of UGT2B17 substrates. Drug companies should include UGT2B17 in early phenotyping assays during drug discovery to avoid late clinical failures.

No abstract

Protein abundance and activity of UGT2B17, a highly variable drug- and androgen-metabolizing enzyme, were quantified in microsomes, S9 fractions, and primary cells isolated from human liver and intestine by validated LC-MS/MS methods. UGT2B17 protein abundance showed >160-fold variation (mean ± SD, 1.7 ± 2.7 pmol/mg microsomal protein) in adult human liver microsomes (n = 26) and significant correlation (r = 0.77, p < 0.001) with testosterone glucuronide (TG) formation. Primary role of UGT2B17 in TG formation compared to UGT2B15 was confirmed by performing activity assays in UGT2B17 gene deletion samples and with a selective UGT2B17 inhibitor, imatinib. Human intestinal microsomes isolated from small intestine (n = 6) showed on average significantly higher protein abundance (7.4 ± 6.6 pmol/mg microsomal protein, p = 0.016) compared to liver microsomes, with an increasing trend towards distal segments of the gastrointestinal (GI) tract. Commercially available pooled microsomes and S9 fractions confirmed greater abundance and activity of UGT2B17 in intestinal fractions compared to liver fractions. To further investigate the quantitative role of UGT2B17 in testosterone metabolism in whole cell system, a targeted metabolomics study was performed in hepatocytes (n = 5) and enterocytes (n = 16). TG was the second most abundant metabolite after androstenedione in both cell systems. Reasonable correlation between UGT2B17 abundance and activity were observed in enterocytes (r = 0.69, p = 0.003), but not in hepatocytes. These observational and mechanistic data will be useful in developing physiologically-based pharmacokinetic (PBPK) models for predicting highly-variable first-pass metabolism of testosterone and other UGT2B17 substrates.

The availability of assays that predict the contribution of cytochrome P450 (CYP) metabolism allows for the design of new chemical entities (NCEs) with minimal oxidative metabolism. These NCEs are often substrates of non-CYP drugmetabolizing enzymes (DMEs), such as UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), carboxylesterases (CESs), and aldehyde oxidase (AO). Nearly 30% of clinically approved drugs are metabolized by non-CYP enzymes. However, knowledge about the differential hepatic versus extrahepatic abundance of non-CYP DMEs is limited. In this study, we detected and quantified the protein abundance of eighteen non-CYP DMEs (AO, CES1 and 2, ten UGTs, and five SULTs) across five different human tissues. AO was most abundantly expressed in the liver and to a lesser extent in the kidney; however, it was not detected in the intestine, heart, or lung. CESs were ubiquitously expressed with CES1 being predominant in the liver, while CES2 was enriched in the small intestine. Consistent with the literature, UGT1A4, UGT2B4, and UGT2B15 demonstrated liver-specific expression, whereas UGT1A10 expression was specific to the intestine. UGT1A1 and UGT1A3 were expressed in both the liver and intestine; UGT1A9 was expressed in the liver and kidney; and UGT2B17 levels were significantly higher in the intestine than in the liver. All five SULTs were detected in the liver and intestine, and SULT1A1 and 1A3 were detected in the lung. Kidney abundance was the most variable among the studied tissues, and overall, high interindividual variability (>15-fold) was observed for UGT2B17, CES2 (intestine), SULT1A1 (liver), UGT1A9, UGT2B7, and CES1 (kidney). These differential tissue abundance data can be integrated into physiologically based pharmacokinetic (PBPK) models for the prediction of non-CYP drug metabolism and toxicity in hepatic and extrahepatic tissues.

Acid ceramidase (AC) is a lysosomal cysteine amidase that controls sphingolipid signaling by lowering the levels of ceramides and concomitantly increasing those of sphingosine and its bioactive metabolite, sphingosine 1-phosphate. In the present study, we evaluated the role of AC-regulated sphingolipid signaling in melanoma. We found that AC expression is markedly elevated in normal human melanocytes and proliferative melanoma cell lines, compared with other skin cells (keratinocytes and fibroblasts) and non-melanoma cancer cells. High AC expression was also observed in biopsies from human subjects with Stage II melanoma. Immunofluorescence studies revealed that the subcellular localization of AC differs between melanocytes (where it is found in both cytosol and nucleus) and melanoma cells (where it is primarily localized to cytosol). In addition to having high AC levels, melanoma cells generate lower amounts of ceramides than normal melanocytes do. This downregulation in ceramide production appears to result from suppression of the de novo biosynthesis pathway. To test whether AC might contribute to melanoma cell proliferation, we blocked AC activity using a new potent (IC 50 ‫؍‬ 12 nM) and stable inhibitor. AC inhibition increased cellular ceramide levels, decreased sphingosine 1-phosphate levels, and acted synergistically with several, albeit not all, antitumoral agents. The results suggest that AC-controlled sphingolipid metabolism may play an important role in the control of melanoma proliferation.Ceramides are a class of bioactive lipids that regulate senescence, apoptosis, and autophagy (1). They comprise more than 200 chemically and functionally distinct molecules and are produced through either de novo biosynthesis or cleavage of preformed sphingolipid precursors in membranes (2-4). They are degraded by the action of five distinct ceramidases, which differ in sequence, structure, subcellular localization, and preferred substrates (5). Among them, acid ceramidase (AC) 2 (also known as N-acetylsphingosine amidohydrolase, ASAH1) is especially relevant due to its possible roles in cancer progression and chemoresistance (6). AC is a lysosomal cysteine amidase that catalyzes the hydrolysis of ceramide into sphingosine and fatty acid with a preference for unsaturated ceramides with 6 -16-carbon acyl chains (5). Its activation is associated with decreased cellular levels of these medium-chain ceramides, which promote senescence and apoptosis, and increased levels of sphingosine 1-phosphate (S1P), which stimulates cell proliferation and cancer cell migration (7). A bioinformatic survey of the National Cancer Institute (NCI) gene expression database conducted in 2003 identified AC as a potential candidate for the development of new biomarkers for the prognosis of melanoma (8). Supporting this possibility, subsequent studies showed that serum of melanoma patients contains AC autoantibodies (9). Moreover, experiments with melanoma cell lines in cultures have demonstrated that dacarbazine, a standard of care for the...